In a previous paper, we demonstrated proteinous chemotactic factors released from hepatocytes exposed to ethanol. In this study, we further characterized the chemotactic factors released from ethanol-treated hepatocytes. After fractionating the conditioned medium with gel chromatography, we demonstrated chemotactic activity at molecular weights of around 20 and 40 kD. When the conditioned medium was mixed with antiserum against interleukin-8, chemotactic activity was almost completely abolished. In addition, antiserum against interleukin-8 completely reduced chemotactic activity of the 20-kD chemotactic factor but did not influence the chemotactic activity of the 40-kD chemotactic factor. After gel electrophoresis of the conditioned medium, the protein reacted with antibody against interleukin-8 was demonstrated at a molecular weight of 20 kD but was not found at a molecular weight of 40 kD. These results suggest that a major part of the proteinous chemotactic factors released from hepatocytes exposed to ethanol could be a dimer form of interleukin-8, one of the proinflammatory cytokines, possibly contributing to the pathogenesis of alcoholic hepatitis.